2.4. Pathologic analysis
Tumors were analyzed in blind conditions by a single pathologist. A representative block of urothelial carcinoma was received from the Anatomic Pathology Service of the University Hospital of Móstoles, and sections stained with hematoxylin and eosin-stained (H&E) were prepared. The H&E sections were reviewed, and the following histological criteria were used to evaluate the tumors:
- Histology of the tumor
The tumor architecture and the type of cells that constitute the tumor were studied. For this purpose, the current WHO 2017 (31) classification parameters of urothelial tumors were used, assessing the following subtypes:
- Urothelial carcinoma with squamous differentiation.
- Urothelial carcinoma with solid nests.
- Papillary urothelial carcinoma.
- Mixed urothelial carcinoma.
- Classification of tumor staging
The classification was conducted according to the “Protocol for the Examination of Specimens From Patients With Carcinoma of the Urinary Bladder” the 8th Edition, AJCC Staging Manual, june 2017 (31) (Table 1).
Table 1
Tumor classification according to the grade of invasion as defined in (31).
Stage
|
Definition
|
Ta
|
Papillary noninvasive carcinoma.
|
Tis
|
Flat urothelial carcinoma in situ.
|
T1
|
Tumor invades lamina propia.
Connective tissue between the urothelium and the detrusor muscle.
|
T2
|
Tumor invades muscularis propria.
Thick bundles of detrusor muscle.
|
T3
|
Tumor invades perivesical tissue.
Adipose tissue beyond muscularis propia.
T3a: microscopic invasion.
T3b: macroscopic invasion.
|
T4
|
Tumor invades other organs.
|
- Histologic grade
The tumors received a single grade of differentiation (high or low) according to the criteria included in the “College of American Pathologist” (31).
- Tumor necrosis or evident apoptosis
Tumors were evaluated for the presence or absence of necrosis. The tumor was classified as negative if the focus of necrosis was less than 10%. Large geographic areas of necrosis (necrotic tumor) were not included.
Samples were classified into two groups: with or without tumor necrosis or apoptosis.
- Presence of nucleolus
The presence or absence of nucleolus in the nuclei was examined. When a nucleolus was identified, it was classified according to its intensity. Samples were classified in three groups defined as follows: G0: nucleolus is not identified or not evident; G+: evident nucleolus is identified at 400x magnification; G++: obvious nucleolus is identified at 200x magnification.
- Growth pattern
The growth pattern of the tumor was examined, and two groups were established: “Growth pattern with exophytic characteristics”, for tumors which show an outward growth, that is, towards the bladder or towards the tubular lumens in the case of tumors located at the level of the ureter or urethra; and “growth pattern of endophytic characteristics”, which included tumors that grow towards the chorion.
- Lymphovascular invasion
The presence of tumor urothelial cellularity inside lymphatic and blood vessels was examined. They were classified into two groups: a first group for tumors in which there is no evidence of invasion in the lymphatic vessels or blood vessels, and a second group in which lymphovascular invasion can be identified.
- Perineural invasion
When studying the nerves, the presence of urothelial carcinoma was observed in different locations, such as within the perineurium, within the nerve, surrounding the nerve or invading ganglionic tissue. Tumors were classified in two groups, according to whether perineural invasion was present or absent.
- Presence of peritumoral lymphocytes
The percentage of lymphocyte cellularity in five high-power fields was analyzed in the peritumoral stroma, preferably considering the areas with more lymphocytes (hot-spot). Tumors were classified in three blocks: Block P0: there is no lymphocytic infiltrate, less than 5%. Block P1: evidence of minimal lymphocytic infiltrate, between 5 and 15%. Block P2: presence of evident lymphocytic infiltrate, greater than 15%.
- Presence of intratumoral lymphocytes
At the tumor level, the percentage of lymphocyte cellularity in five high-magnification fields of each sample was studied, preferably considering the areas that had more lymphocytes (hot-spot). Apoptotic cells were not counted. Classification was designed in three blocks: Block L0: there is no lymphocytic infiltrate, less than 5%; Block L1: evidence of minimal lymphocytic infiltrate, between 5 and 15%; Block L2: presence of evident lymphocytic infiltrate, greater than 15%.